Nucleosides and nucleotides (nucleos(t)ides) have been in clinical use for almost 50 years and have become cornerstones of treatment for patients with viral infections or cancer. The approval of several additional drugs over the past decade demonstrates that this family still possesses strong potential. Therefore nucleos(t)ide are of great interest as promising chemotherapeutic agents, including: 2′-deoxy-1-uridine (CAS  31501-19-6), 2′-deoxy-D-uridine (CAS  951-78-0), telbivudine (CAS  3424-98-4), zidovudine (AZT, CAS  30516-87-1), trifluridine (CAS  70-00-8), clevudine (CAS  163252-36-6), PSI-6206 (CAS  863329-66-2), 2′-(S)-2′-chloro-2′-deoxy-2′-fluorouridine (CAS  1673560-41-2), ND06954 (CAS  114248-23-6), stavudine (CAS  3056-17-5), 5-ethynyltavudine (Festinavir, CAS  634907-30-5), torcitabine (CAS  40093-94-5), (−)-beta-D-(2R,4R)-dioxolane-thymine (DOT, 1-((2R,4R)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)-5-methyl-2,4 (1H,3H)-pyrimidinedione, CAS  127658-07-5), 2-(6-amino-purin-9-yl)-ethanol (CAS  707-99-3), 2′-C-methylcytidine (CAS  20724-73-6), PSI-6130 (CAS  817204-33-4), gemcitabine (CAS  95058-81-4), 2′-chloro-2′-deoxy-2′-fluorocytidine (CAS  1786426-19-4), 2′,2′-dichloro-2′-deoxycytidine (CAS  1703785-65-2), 2′-C-methylcytidine (CAS  20724-73-6), PSI-6130 (CAS  817204-33-4), lamivudine (3TC, CAS  134678-17-4), emtricitabine (CAS  143491-57-0), 2′-deoxyadenosine (CAS  958-09-8), 2′-deoxy-β-L-adenosine (CAS  14365-45-8), 2′-deoxy-4′-C-ethynyl-2-fluoroadenosine (CAS  865363-93-5), didanosine (CAS  69655-05-6), entecavir (CAS  209216-23-9), FMCA (CAS  1307273-70-6), dioxolane-G (DOG, CAS  145514-01-8), β-D-2′-deoxy-2′-(R)-fluoro-2′-β-C-methylguanosine (CAS  817204-45-8), abacavir (ABC, CAS  136470-78-5), dioxolane-A (DOA, CAS #145514-02-9), [(2R,4R)-4-(6-cyclopropylamino-purin-9-yl)-[1,3]dioxolan-2-yl]-methanol (CAS  1446751-04-7), amdoxovir (AMDX, CAS  145514-04-1), (R)-1-(6-amino-purin-9-yl)-propan-2-ol (CAS  14047-28-0), and [(2S,5R)-5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yl]-methanol [M. J. Sofia. Nucleosides and Nucleotides for the treatment of viral diseases. In Annual Reports in Medicinal Chemistry 2014, Volume 49, Editor-in-Chief M. C. Desai, p 221-247. L. P. Jordheim et al. Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug. Discov. 2013, 12(6), 447-464.].
Currently, nucleos(t)ides are the preferred option and standard of care for treating patients infected with hepatitis B virus (HBV) and they are emerging as a key component in therapies to treat hepatitis C virus (HCV) infection. They also play a central role in the management of other viral infections such as those caused by herpes viruses (HSV-1 and HSV-2), varicella zoster virus, Epstein-Barr virus, and cytomegalovirus [E. De Clercg. Ed. Antiviral Agents 2013, Vol. 67: Academic Press: New York. 2013. L. P. Jordheim et al. Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nal. Rev. 2013,12, 447-464.]. The attractiveness of a nucleos(t)ide strategy in the development of therapeutics for vital diseases sterns from the fact that all viruses require a polymerase for either DNA or RNA replication.
Another factor that must be considered when developing a nucleos(t)ide inhibitor pertains to nucleos(t)ide metabolic activation. It is the nucleotide triphosphate analog, as the functional substrate for the viral polymerase that becomes incorporated into the growing RNA or DNA chain, typically leading to a chain termination event and ultimately an end to viral replication. Consequently, the efficiency by which a nucleos(t)ide gets converted to the active triphosphate and the concentration and half-life of the triphosphate within the cell are important factors in how effective the nucleos(t)ide is as an inhibitor of viral replication. In general, the first phosphorylation step is the most discriminating among the three needed to generate the active triphosphate. In cases where the nucleoside itself is not a good substrate for the kinase involved in the initial phosphorylation step, delivery of the monophosphate is desired, but this typically requires the use of prodrug technology to mask the unfavorable characteristics of the phosphate group and facilitate permeability. Consequently, nucleotide prodrug strategies have seen much use in the development of nucleotides to treat viral and cancer diseases.
Recently, the uridine nucleotide prodrug Sovaldi® (sofosbuvir, PSI-7977; GS-7977) [M. J. Sofia et al. Discovery of a β-D-20-Deoxy-20-r-fluoro-20-β-C-methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C Virus. J Med. Chem. 2010, 53, 7202-7218. M. J. Sofia et al. Nucleoside phosphoramidate prodrugs. U.S. Pat. No. 7,964,580 (2011).] became the first nucleos(t)ide approved by both the FDA and EU regulatory authorities for the treatment of HCV patients infected with genotype (gT) 1, 2, 3, and 4 HCV virus and in clinical trials it also showed efficacy against all relevant HCV gTs (1-6) [I. M. Jacobson et al. Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. Engl. J. Med. 2013, 368, 1867-1877. E. Lewirz et al. Sofosbuvir for previously untreated chronic hepatitis C infection. Engl. J. Med. 2013, 368, 1878-1887]. Its approval marked the first introduction of an all-oral interferon (IFN)-free regimen to treat patients suffering from HCV infection.
Also other known chemotherapeutic agents include phosphoramidate moieties to treat hepatitis C, including AVI-4201 [A. V. Ivachtchenko et al. Alkyl 2-{[(2r, 3s, 5r)-5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-3-hydroxy-tetrahydro-furan-2-yl-methoxy]-phenoxy-phosphoryl-amino}-propionates, nucleoside inhibitors of HCV NSSB RNA-polymerase, and methods for producing and use thereof. WO2014148949, 2014], AVI-4203 [A. V. Ivachtchenko et al. Substituted (S)-(2R, 3R, 5R)-3-hydroxy-(5-pyrimidin-1-yl)-tetrahydrofuran-2-ylmethyl aryl phosphoramidate. U.S. Pat. No. 8,889,701, 2014] or CC-1845 [D. L Mayers. Development of Potent Novel Oral Pan-genotypic HCV Nucleotide, NS5A, NS5B non-nucleoside, and Helicase Inhibitors. 2015. https://www.informedhorizons.com/hepdart2015/pdf/Presentations/Mayers.pdf].

Should be noted that in this case the inhibitory activity of HCV NS5B RNA-polymerase phosphoramidate inhibitors Sovaldi®, AVI-4201 AVI-4203 ℏ CC-1845 by orders of magnitude higher than that of the corresponding nucleoside: PSI-6206, Gemcitabine and 2′-C-Methylcytidine.
Other guanosine nucleotide prodrugs have been investigated, also employing the phosphoramidate prodrug moiety in an attempt to leverage liver targeting. Thus, PSI-353661 demonstrated potent inhibition in the replicon assay (EC90=0.008 μM→1000-fold increase in potency compared to the guanosine analogue-β-D-2′-deoxy-2′-R-fluoro-2′-β-C-methylguanosine (Table 1).) and a novel resistance profile similar to PSI-352938, but was never progressed into clinical development [W. Clung et al. Discovery of PSI-353661, a Novel Purine Nucleotide. ACS Med. Chem. Lett. 2011. 2. 130-135.]. The structurally related pro-drugs IDX-184 (EC50=0.4 μM) [X.-J. Zhou. Et al. Safety and Pharmacokinetics of IDX184, a Liver-Targeted Nucleotide Polymerase Inhibitor of Hepatitis C Virus, in Healthy Subjects. Antimicrob. Agents Chemother. 2011, 55, 76-81. J. Lalezari, et al. Short-Term Monotherapy with IDX184, a Liver-Targeted Nucleotide Polymerase Inhibitor, in Patients with Chronic Hepatitis C Virus Infection. Antimicrob. Agents Chemother. 2012. 56, 6372-6378.] and INX-08189 (BMS-986094, EC50=0.010 μM) [C. McGuigan et al. Phosphorodiamidates as a Promising New Phosphate Prodrug Motif for Antiviral Drug Discovery: Application to Anti-HCV Agents. J. Med. Chem. 2011, 54, 8632-8645. J. H. Vernachio et al. MX-08189, a phosphoramidate prodrug of 6-O-methyl-2′-C-methyl guanosine, is a potent inhibitor of hepatitis C virus replication with excellent pharmacokinetic and pharmacodynamic properties. Antimicrob. Agents Chemother. 2011. 55, 1843-1851.], each producing an identical triphosphate, were progressed into the clinic, but severe cardiovascular toxicity associated with INX-08189 resulted in discontinuation of development for both compounds [J. J. Arnold et al. Sensitivity of Mitochondrial Transcription and Resistance of RNA Polymerase II Dependent Nuclear Transcription to Antiviral Ribonucleosides. PLOS Pathog. 2012. 8, DOI: 10.1371/journal.ppat. 1003030.]. The severe nature of the cardiovascular toxicity seen with INX-08189 seems to have curtailed the interest in developing a guanosine nucleoside for treating HCV patients.

HBV is a DNA virus in the Hepadnaviridae family. It is estimated that 400 million individuals are infected with HBV worldwide. The current standard of care for treatment of HBV is long-term nucleos(t)ide therapy. The nucleos(t)ides approved for treating HBV infection include lamivudine, adefovir dipivoxil, entecavir, telbivudine, and TDF. Entecavir and TDF are the most widely prescribed of these agents. Long-term use of entecavir leads to resistance in a significant patient population and TDF is associated with nephrotoxicity and bone loss [D. Grimm et al. HBV life cycle and novel drug targets. Hepatol. Int. 2011. 5. 644-653. G. Borgia, I. Gentile. Treating chronic hepatitis B: today and tomorrow. Curr. Med. Chem. 2006. 13. 2839-2855.]. However, continued use of nucleos(t)ide therapy has been associated with reduction in liver fibrosis demonstrating that suppression of viral replication has positive long-term value [T. T. Chang et al. Long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis B. Hepatology 2010. 52, 886-893. P. Marcellin et al. Regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis B: a 5-year open-label follow-up study. Lancet 2013, 381, 468-475.].
Even with the success of existing nucleos(t)ide HBV therapy, work has continued in an effort to identify, novel inhibitors that may provide additional benefit relative to the existing agents, and several of the anti-HW agents mentioned above have also been assessed for us in treating HBV infection [C. A. Geng et al. Small-molecule inhibitors for the treatment of hepatitis B virus documented in patents. Mini Rev. Med. Chem. 2013. 13, 749-776.].
Recently, preparation of the 2′-fluoro-6′-methylene-carbocyclic adenosine (FMCA) (EC50=0.55 μM), which borrowed the 6′-methylene-carbocyclic nucleus of entecavir, led to a potent inhibitor of HBV replication that was also active against the lamivudine-entecavirresistant clone (L180M+M204V+S202G) [R. K. Rawal et al. 2′-Fluoro-6′-methylene-carbocyclic adenosine phosphoramidate (FMCAP) prodrug: In vitro anti-HBV activity against the lamivudineentecavir resistant triple mutant and its mechanism of action. Bioorg. Med. Chem. Lett. 2013. 23, 503-506.]. Furthermore, preparation of the corresponding 5′-phosphoramidate of FMCA resulted in a compound that was 10-fold more potent than FMCA against both the wild-type (EC50=0.62 μM) and resistant mutant (EC50=0.054 μM) [R. K. Rawal et al. 2′-Fluoro-6′-methylene-carbocyclic adenosine phosphoramidate (FMCAP) prodrug: In vitro anti-HBV activity against the lamivudineentecavir resistant triple mutant and its mechanism of action. Bioorg. Med. Chem. Lett. 2013. 23, 503-506.].
It is also known that the phosphoramidate conjugates of clevudine (EIDD-02173) retained potent anti-HBV activity in cell culture models of infection. The phosphoramidate moiety successfully delivered clevudine-5′-monophosphate to the liver while significantly decreasing non-liver organ exposure. Selective targeting of the liver could potentially lead to a decrease in the off-target effects related to clevudine in humans. [G. R. Bluemling et al. Targeted Delivery of Clevudine-5′-Monophosphate to the Liver After Oral Administration of a Clevudine-5′-Phosphoramidate Conjugate to Rats for the Treatment of HBV Infections. Global Antiviral Journal 2015, 11, Suppl. 3: HEP DART 2015: Abstr. 104, P. 97].

Gemcitabin-5′-phosphoramidate (NUC-1031) [M. Slusarczyk et al. Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development. J. Med. Chem. 2014, 57, 1531-1542] showed a high anti-cancer activity. In particular NUC-1031 significantly reduced tumor volume in vivo in xenografts models of human pancreatic cancer. Important to note that activation of NUC-1031 is much less dependent on the nucleoside transporters and deoxycytidine than gemcitabine. In addition, NUC-1031 is resistant to cytidine deaminase degradation unlike gemcitabine.

It should be noted that the structure of phosphoramidite moiety has a significant impact on the stability of phosphoramidate nucleosides in various media, their pharmacokinetics, bioavailability, distribution in body organs and the selectivity of their action [M. J. Sofia et al. 2010. P. Wang et al. Phosphoramidate prodrugs of (−)-β-D-(2R, 4R)-dioxolane-thymine (DOT) as potent anti-HIV agents. Antiviral Chem. Chemotherapy 2012, 22, 217-238. L. Bondada et al. Adenosine Dioxolane Nucleoside Phosphoramidates as Antiviral Agents for Human Immunodeficiency and Hepatitis B Viruses. ACS Med. Chem. Lett. 2013, 4, 747-751. M. Slusarczyk et al. Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development. J. Med. Chem. 2014, 57, 1531-1542.].
So far synthesis of new phosphoramidate nucleosides and their use as chemotherapeutic agents for the treatment of viral diseases and cancer are highly relevant.
Is important to note also that until now nucleoside-containing macroheterocyclic phosphoramidates and their use for treating viral and cancerous diseases were unknown.